The present invention relates to a process for the direct cryo-preservation of plant tissues wherein the tissues are primary explants that have been subjected to an induction treatment for regeneration.
It is widely acknowledged that for long-term storage of genetic plant material, such as of germplasm of vegetatively propagated species and of recalcitrant species, cryo-preservation is the desired option. This process involves storage at ultra-low temperatures, usually that of liquid nitrogen (xe2x88x92196xc2x0 C.), at which temperature cell division and metabolic processes come to a stop and the plant material can thus be stored without modification or alteration for prolonged periods of time. Other advantages of this technique reside in that cryo-preserved material requires only limited space, is protected from contamination and needs only moderate maintenance efforts.
Processes for the long-term preservation of plants have been developed only for a restricted number of plant species, wherein the original plant or parts thereof are treated in a number of steps so as to obtain explants in developmental late stages including zygotic and somatic embryos, meristems, etc., which are found to be eventually capable to grow to a new plant after cryo-preservation. However, it is acknowledged that the longer the duration of the in vitro culture before cryo-preservation, that is the higher the number of pretreatment steps, the higher the risk of somaclonal variation and the bigger the amount of laboratory work to prepare the samples to be preserved by freezing. In consequence, said processes may not be applied to a large collection of plant genetic material.
It would therefore be desirable to limit the steps in culture before cryo-preservation to as few as possible.
Apart from the drawbacks involved in subjecting the plant derived tissue to a number of pretreatment steps to arrive at the desired explant tissue that is finally capable to regenerate to a new plant, some developmental stages of the plant, such as, e.g., zygotic embryos, are found to be merely usable for pure lines of self-pollinated species thus limiting the applicability of such procedures. For other species merely vegetative tissues may be used, such as meristems, shoot tips, regenerating tissues or somatic embryos. Consequently, the choice of the specific material for storage will depend on a variety of parameters, such as the plant species, the techniques available, the genetic structure of the plant being stored and the available germplasm.
Finally, in order to limit the risk of somaclonal variation, the preserved tissue should regenerate from the cryo-preserved material as directly as possible into plantlets. Therefore, calli and/or embryogenic tissues are less desirable than somatic embryos, which could directly regrow without any secondary embryogenesis or callogenesis.
The present invention provides a method for cryo-genically storing any one of a wide variety of different plant species without involving the laborious steps of culturing plant material until a later developmental stage of the plant is obtained, ie., one that is capable to grow to a full plant. Thus, different plants may be processed for storage at low temperatures without involving complicated steps to obtain a plant tissue suitable for regeneration to a new plant. This process advantageously includes the steps of dehydrating, pre-freezing and cryo-freezing plant derived tissue, wherein the plant tissue to be cryo-preserved is a primary explant that has been subjected to an induction treatment for regeneration.
In a preferred embodiment, the plant tissue utilized in the process according to the present invention is a primary explant capable to regenerate buds. In another preferred embodiment the process for the cryo-preservation of the present invention comprises using a plant tissue capable to regenerate embryos.